Team:Washington/Celiacs/Methods

From 2011.igem.org

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=Redesigning Kumamolisin to Have Higher Activity at Low pH=
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<center><big><big><big><big>'''Gluten Destruction: Methods'''</big></big></big></big></center><br><br>
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[[File:Washington Foldit.png|400px|thumb|right|A Sample Mutation in Foldit Showing a Change from Glycine to Serine]]
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==Using Foldit to Design Mutations==
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='''Redesigning Kumamolisin to Have Higher Activity at Low pH'''=
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In order to design mutations to wild-type Kumamolisin that would increase the enzyme’s proteolytic activity on gluten, we used a computational enzyme editing program called Foldit, which allows the user to hypothetically modify the amino acid sequence of a protein by creating point mutations at any location within the protein’s crystal structure. Within Foldit, we loaded Kumamolisin’s crystal structure in complex with a model PQLP peptide that recurs frequently in gluten, thus mimicking gluten as a substrate. We then modified the amino acid residues around the active site of Kumamolisin in the crystal structure, attempting to decrease the free energy of, and thus stabilize, the system, based on estimations of free energy from algorithms run by Foldit.
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[[File:Washington Foldit.png|550px|thumb|right|A Sample Mutation in [http://fold.it Foldit] Showing a Change from Glycine to Serine]]
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=='''Using [http://fold.it Foldit] to Design Mutations'''==
 +
In order to design mutations to wild-type Kumamolisin that would increase the enzyme’s proteolytic activity on gluten, we used a computational enzyme editing program called [http://fold.it Foldit], which allows the user to hypothetically modify the amino acid sequence of a protein by creating point mutations at any location within the protein’s crystal structure.  
 +
 
 +
Within Foldit, we loaded Kumamolisin’s crystal structure in complex with a model PQLP peptide that recurs frequently in gluten, thus mimicking gluten as a substrate. We then modified the amino acid residues around the active site of Kumamolisin in the crystal structure, attempting to decrease the free energy of, and thus stabilize, the system. Estimations of free energy were based on algorithms run by Foldit.
Using this method, we designed over 100 novel mutants, each of which could potentially increase Kumamolisin’s proteolytic activity on gluten.
Using this method, we designed over 100 novel mutants, each of which could potentially increase Kumamolisin’s proteolytic activity on gluten.
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==Mutagenizing Kumamolisin==
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----
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='''Mutagenizing Kumamolisin'''=
 +
 
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Kunkel mutagenesis is a classic procedure for incorporating targeted mutations into a piece of DNA, so it was ideal for changing our wild-type Kumamolisin gene to code instead for specifically designed variant enzymes.
[[File:Washington Kunkels.png|500px|thumb|left|Overview of how Kunkel Mutagenesis works]]
[[File:Washington Kunkels.png|500px|thumb|left|Overview of how Kunkel Mutagenesis works]]
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===Producing ssDNA===
 
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Once we had obtained feasible mutants from the Foldit program, we ordered these mutations in oligonucleotide primers to be used in Kunkel Mutagenesis. Kunkel mutagenesis is a short and simple procedure, so it was ideal for producing many mutants at a time To perform the mutagenesis, we first transformed the plaasmid containing wild-type kumamolisin into chemically competent cells of ''Escherichia coli'' CJ236. These cells were then grown and harvested for their single stranded DNA (ssDNA). We then determined the concentration of the DNA using a nanodrop machine. This was done to ensure that the DNA was at a high enough concentration to be used for mutagenesis.
 
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===Kunkel Mutagenesis===
 
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Using ssDNA as a template, we annealed oligo nucleotides in a PCR block to increase specific binding. The primer was then extended on the ssDNA using T7 DNA polymerase. We then dialyzed the DNA to remove salts introduced in previous steps. We then added the DNA to cuvettes containing electrically competent ''E. coli'' BL21* cells. These cells were then pulsed with electricity to allow the uptake of the newly mutagenized DNA. After growing the cells for an hour in TB, we plated the cells on LB agar containing kanamycin.
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=='''Kunkel Mutagenesis'''==
 +
 
 +
The first step to producing our specially designed enzymes was to change the wild-type gene that codes for Kumamolisin to code instead for variant enzymes with our desired amino acid substitutions.  
 +
 
 +
We designed mutagenic oligonucleotide primers that would anneal to the wild-type Kumamolisin gene and incorporate point mutations that, when expressed, would result in a variant of Kumamolisin with the desired amino acid shift.
 +
 
 +
To incorporate these mutations, we first isolated single stranded DNA (ssDNA) of our vector harboring the wild-type Kumamolisin gene. To do this we infected cells with bacteriophage M13, which packages its own ssDNA genome identified by length, and so in tandem packaged our vector in single stranded form. We then harvested the phage from the lysed culture of E. coli, and extracted our single stranded vector DNA.
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 +
Next, we annealed and extended our mutagenic oligos to incorporate the specified mutations into the newly synthesized antisense strand. This hybrid vector was transformed into E. coli that degraded the original uracil-containing DNA and replaced it with sections complementary to the mutagenized strand.
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 +
 
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='''Using a Whole Cell Lysate Assay to Test Activity of Mutants'''=
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To test our designs, we developed a whole cell lysate assay that allowed us to perform a rough screen of a large number of mutants. In this assay, we expressed our mutant enzymes in <i>E. coli</i>, lysed the cells and separated the enzymes from large cell particulate. We then performed the assay at pH 4, mimicking the gastric environment. We added our model PQLP peptide, conjugated to both a fluorophore and a quencher so that no fluorescence would be achieved until after the peptide had been enzymatically cleaved. We then measured the fluorescence of each reaction at 30 second intervals, and were thereby able to estimate relative activity on breaking down PQLP by increase in fluorescence of the system.
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[[File:Washington Whole Cell Lysate Assay.jpg|center|General Overview of the Whole Cell Lysate]]
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='''Testing Purified Mutants to Accurately Assess Activity'''=
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[[File:Washington First Raw Data.png|right|500px|thumb|We measured fluorescence of each reaction at 30 second intervals to see the rate at which each mutant cleaved PQLP.]]
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=='''Purification'''==
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From our whole cell lysate screen of each design, we identified mutants that showed the most increase in activity from the wild-type Kumamolisin. We then proceeded to purify these most promising variants and test them against the wild-type and against SC PEP using the same fluorescence metric designed for the whole cell lysate assay. The key difference between the whole cell assay and the purified protein assay is that in the latter we were able to control the concentration of enzyme in each well, adjusting for the possibility of varying expression levels and thus enzyme concentrations in the whole cell lysate assay.
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==Using a Whole Cell Lysate Assay to Test Activity of Mutants==
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Purification was performed via Nickel-affinity chromatography, and resulting protein concentrations were measured using ultraviolet-visible spectrophotometry.
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After the cells had been allowed to grow overnight, colonies were picked and used to inoculate a 96 well plate containing LB and kanamycin. This step allowed us to grow a representative amount of cells containing each mutation. After growing overnight at 37 degrees celcius cells from each well were transferred to another 96 well plate containing TB and kanamycin. These plates were incubated at 37 degrees celcius and later induced using IPTG. After induction, we incubated the plates at 18 degrees celcius overnight. We then lysed the cells and tested the supernatant for proteolytic activity towards PQLP in an assay which measured PQLP degradation over a period of 30 minutes. The assay was done at pH 4 in accordance with the assays done to test ScPEP according to the literature. The mutants were tested against wild-type kumamolisin and ScPEP, an enzyme currently used for the treatment of gluten intolerance via proteolysis. The assay we used was not highly accurate in terms of actual activity. However, what the assay allowed us to do was determine activity relative to our controls. This allowed us to determine which mutants were worth purifying to get more accurate activity data.
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[[File:Washington Assay.png|center|General Overview of the Whole Cell Lysate]]
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==Testing Purified Mutants to Accurately Assess Activity==
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=='''Assay'''==
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===Purification===
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Concentration dependent assays were performed for each promising mutant. We measured the fluorescence of each reaction at 30 second intervals to see the rate at which fluorescence increased, thus obtaining a relative rate of cleavage of PQLP by increase in fluorescence of the system. Raw data appeared as shown right, and the slope of each line was calculated, giving us relative rate information that could be used in conjunction with rate information obtained in the same assay for native Kumamolisin to determine fold change in activity.
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After compiling a set of mutants which showed a relative increase in activity we proceeded to purify our mutant proteins. This step is crucial because it allows us to determine how our mutant compares with the wild-type on a quantitative level. For instance, if the whole cell lysate assay showed one mutant to have ten times the activity level as wild-type kumamolisin we cannot assume that there has been an increase in activity because there could simply be ten times the amount of protein with the same level of activity. To purify our mutants, we first grew them in TB and kanamycin with a single colony of the mutant. This inoculation was grown over 24 hours at 37 degrees celcius and then expanded to 50 mililiters (TB+kanamycin). We then allowed the culture to grow to a specific optical density of cells before inducing the culture using IPTG. We then allowed the cells to grow again to express kumamolisin. We then lysed the cells using a lysis buffer and centrifugation. This allowed the enzymes to be released from the cells. The proteins were then collected on a column, washed with buffer, and eluted off the column. Finally, wwe dialyzed the protein in a sodium acetate buffer (pH 4).
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===Assay===
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Once we had pure protein, we determined the concentration of each using a NanoDrop machine. With the concentration of each, we then diluted the proteins to the same concentration. For the assay, we also used purified wild-type kumamolisin and ScPEP. The assay was again run for 30 minutes at pH 4. Using the data from the purified assays, we were able to deermine which mutants had higher activity than kumamolisin and by how much their activity was  greater.
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Latest revision as of 02:36, 29 September 2011


Gluten Destruction: Methods


Redesigning Kumamolisin to Have Higher Activity at Low pH

A Sample Mutation in [http://fold.it Foldit] Showing a Change from Glycine to Serine

Using [http://fold.it Foldit] to Design Mutations

In order to design mutations to wild-type Kumamolisin that would increase the enzyme’s proteolytic activity on gluten, we used a computational enzyme editing program called [http://fold.it Foldit], which allows the user to hypothetically modify the amino acid sequence of a protein by creating point mutations at any location within the protein’s crystal structure.

Within Foldit, we loaded Kumamolisin’s crystal structure in complex with a model PQLP peptide that recurs frequently in gluten, thus mimicking gluten as a substrate. We then modified the amino acid residues around the active site of Kumamolisin in the crystal structure, attempting to decrease the free energy of, and thus stabilize, the system. Estimations of free energy were based on algorithms run by Foldit.

Using this method, we designed over 100 novel mutants, each of which could potentially increase Kumamolisin’s proteolytic activity on gluten.


Mutagenizing Kumamolisin

Kunkel mutagenesis is a classic procedure for incorporating targeted mutations into a piece of DNA, so it was ideal for changing our wild-type Kumamolisin gene to code instead for specifically designed variant enzymes.

Overview of how Kunkel Mutagenesis works




Kunkel Mutagenesis

The first step to producing our specially designed enzymes was to change the wild-type gene that codes for Kumamolisin to code instead for variant enzymes with our desired amino acid substitutions.

We designed mutagenic oligonucleotide primers that would anneal to the wild-type Kumamolisin gene and incorporate point mutations that, when expressed, would result in a variant of Kumamolisin with the desired amino acid shift.

To incorporate these mutations, we first isolated single stranded DNA (ssDNA) of our vector harboring the wild-type Kumamolisin gene. To do this we infected cells with bacteriophage M13, which packages its own ssDNA genome identified by length, and so in tandem packaged our vector in single stranded form. We then harvested the phage from the lysed culture of E. coli, and extracted our single stranded vector DNA.

Next, we annealed and extended our mutagenic oligos to incorporate the specified mutations into the newly synthesized antisense strand. This hybrid vector was transformed into E. coli that degraded the original uracil-containing DNA and replaced it with sections complementary to the mutagenized strand.







Using a Whole Cell Lysate Assay to Test Activity of Mutants

To test our designs, we developed a whole cell lysate assay that allowed us to perform a rough screen of a large number of mutants. In this assay, we expressed our mutant enzymes in E. coli, lysed the cells and separated the enzymes from large cell particulate. We then performed the assay at pH 4, mimicking the gastric environment. We added our model PQLP peptide, conjugated to both a fluorophore and a quencher so that no fluorescence would be achieved until after the peptide had been enzymatically cleaved. We then measured the fluorescence of each reaction at 30 second intervals, and were thereby able to estimate relative activity on breaking down PQLP by increase in fluorescence of the system.

General Overview of the Whole Cell Lysate



Testing Purified Mutants to Accurately Assess Activity

We measured fluorescence of each reaction at 30 second intervals to see the rate at which each mutant cleaved PQLP.

Purification

From our whole cell lysate screen of each design, we identified mutants that showed the most increase in activity from the wild-type Kumamolisin. We then proceeded to purify these most promising variants and test them against the wild-type and against SC PEP using the same fluorescence metric designed for the whole cell lysate assay. The key difference between the whole cell assay and the purified protein assay is that in the latter we were able to control the concentration of enzyme in each well, adjusting for the possibility of varying expression levels and thus enzyme concentrations in the whole cell lysate assay.

Purification was performed via Nickel-affinity chromatography, and resulting protein concentrations were measured using ultraviolet-visible spectrophotometry.

Assay

Concentration dependent assays were performed for each promising mutant. We measured the fluorescence of each reaction at 30 second intervals to see the rate at which fluorescence increased, thus obtaining a relative rate of cleavage of PQLP by increase in fluorescence of the system. Raw data appeared as shown right, and the slope of each line was calculated, giving us relative rate information that could be used in conjunction with rate information obtained in the same assay for native Kumamolisin to determine fold change in activity.

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